Kazushige Yokoyama

Analysis of the nucleotide sequence of chromosome VI from Saccharomyces cerevisiae.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VI (270 kb) has revealed that it contains 129 predicted or known genes (300 bp or longer). Thirty–seven (28%) of which have been identified previously. Among the 92 novel genes, 39 are highly homologous to previously identified genes. Local sequence motifs were compared to active ARS regions and inactive loci with perfect ARS core sequences to examine the relationship between these motifs and ARS activity. Additional ARS sequences were predominantly observed in 3′ flanking sequences of active ARS loci.

Phosphorylation of the adenovirus E1A-associated 300 kDa protein in response to retinoic acid and E1A during the differentiation of F9 cells.

Transcription of the c‐jun gene is up‐regulated by either retinoic acid (RA) or adenovirus E1A during the differentiation of F9 cells. We show here that RA and E1A induce phosphorylation of the E1A‐associated 300 kDa protein (p300) during the differentiation of F9 cells. The region of E1A that is required for interaction with cellular protein p300 overlaps with the region of E1A required for E1A to induce expression of the c‐jun gene. Treatment of F9 cells with RA or infection of the cells by adenovirus led to a decrease in the electrophoretic mobility of p300. Phosphatase treatment of p300 from RA‐treated or adenovirus‐infected F9 cells reversed the changes in migration of p300, indicating that RA‐ and E1A‐mediated changes in the mobility of p300 were due to phosphorylation. We also found factors, designated DRF1 and DRF2, that bound specifically to a sequence element that is necessary and sufficient for RA‐ and E1A‐mediated up‐regulation of the c‐jun gene. The mobility of DRF complexes was changed by E1A or RA and the complexes were supershifted by addition of a polyclonal p300 antiserum. Moreover, overexpression of p300 resulted in an increase in the level of DRF1 complex. p300 fused to the DNA binding domain of the E2 protein of papilloma virus stimulated E2‐dependent reporter activity in response to RA or E1A in F9 cells. Our results suggest that p300 is part of the DRF complexes, that it is differentially phosphorylated in undifferentiated versus differentiated cells and that it is likely involved in regulating transcription of the c‐jun gene during F9 cell differentiation.

Transcriptional regulation of the c-jun gene by retinoic acid and E1A during differentiation of F9 cells.

Differentiation of mouse F9 embryonal carcinoma (EC) cells can be induced by exposure to retinoic acid (RA) or by expression of adenovirus E1A. The transcription of the c‐jun gene is stimulated by either RA or E1A. We report here that both RA and E1A strongly induce the expression of chloramphenicol acetyltransferase (CAT) from c‐jun promoter/CAT reporter construct (c‐jun/CAT), which is stably integrated into F9 cells, in a manner that is independent of both copy number and integration locus. The induction of c‐jun/CAT expression is observed in undifferentiated F9 cells, but not in differentiated F9 cells, adenovirus‐infected F9 cells or HeLa cells. Deletion analysis of the promoter region of the c‐jun gene indicates that the sequence elements required for the RA‐ and E1A‐mediated induction are identical and they have been defined as a region of 145 bp between −190 and −46 of the 5′ flanking region of c‐jun. This RA and E1A response element (RERE) contains five variants of the motif CGCGGTGACGNT. The upstream two motifs are adjacent and extend in opposite directions, creating an imperfect palindrome. The downstream four motifs are located at 35 or 36 bp intervals in the same orientation. Substitution and insertion analysis indicates that these motifs and their regular intervals are important for the activity of the RERE.

Cloning of a new kinesin-related gene located at the centromeric end of the human MHC region

We previously reported the presence of a new gene (HSET) with an unknown function, in the centromeric side of the class II gene region of the human major histocompatibility complex (MHC). cDNA clones corresponding to the HSET gene were isolated from a human testis cDNA library. A 2.4 kilobase transcript from the HSET gene was abundantly expressed in testis, B-cell, T-cell, and ovary cell lines but was not detected in lung or stomach. Analysis of the nucleotide sequence of the HSET cDNA clones revealed significant similarity to kinesin-related proteins in yeast, Drosophila, and human. Its predicted amino acid sequence contains a domain with strong sequence similarity to the ATP-binding and motor domains of a plus end-directed microtuble motor protein, kinesin, which might be involved in mitotic chromosome segregation, suggesting that the HSET gene encodes a novel kinesin-related protein.

Mapping of the gene family for human heat-shock protein 90α to chromosomes 1, 4, 11, and 14

Abstract The HSP90 family of heat-shock proteins (encoded by genes for HSP90α and β) constitutes one of the major groups of proteins that are synthesized at increased rates in response to heat and other forms of stress. We previously isolated two distinct cDNA clones for HSP90α from human peripheral blood lymphocytes and from HeLa cells transfected with the adenovirus E1A gene, respectively. To determine the organization of this complex multigene family in the human genome, we used three complementary approaches: Southern analysis of a panel of human/hamster somatic cell hybrids, molecular cloning of the cosmid HSP90α clones from libraries prepared with DNAs from human lymphoblastoid cells, and in situ hybridization to human chromosomes. We demonstrate here that nucleotide sequences that encode HSP90α map to human chromosomes 1q21.2–q22, 4q35, 11p14.1–p14.2, and 14q32.3. The chromosomal mapping of the loci, HSPCAL1, HSPCAL2, HSPCAL3, HSPCAL4, and the characterization of the respective genes should facilitate clarification of the organization of this gene family and lead to a better understanding of the biological functions of the gene product.

Analysis of YAC clones by pulsed-field gel electrophoresis: Physical mapping of Cu/Zn superoxide dismutase gene locus

Procedures for constructing a physical map of yeast artificial chromosome (YAC) clones by pulsed-field gel electrophoresis are described. Included are practical protocols for preparation of yeast chromosomal DNA containing YAC in agarose plugs, restriction enzyme digestion of DNA within the plugs, and running conditions for pulsed-field gel electrophoresis and Southern hybridization. As an illustration, we have applied these methods to two independent YAC clones encoding the Cu/Zn superoxide dismutase gene (SOD-1) and constructed from these two overlapping clones a restriction enzyme map spanning approximately 600 kb. The map of chromosome 21q22 may prove useful in investigating the genetic basis of Down syndrome.

A novel pathway for retinoic acid-induced differentiation of F9 cells that is distinct from receptor-mediatedTrans-activation

SummaryRetinoic acid (RA) has striking effects on vertebrate development and induces differentiation of several lines of cells including embryonal carcinoma F9 cells. It is generally accepted that the actions of RA are mediated by nuclear receptors for RA. However, we now provide evidence that F9 cells can differentiate in response to RA withouttrans-activation by nuclear receptors. Irreversible differentiation of F9 cells was induced by 18 h of exposure of RA with subsequent incubation in the absence of RA. This induction of differentiation was not blocked after inhibition of protein synthesis and mRNA synthesis during the 18-h treatment with RA, but the endogenous RA receptors failed to activate transcription from their target genes that contain the receptor-binding sequences. During the commitment of RA-induced differentiation, at least five sets of four phosphorylated proteins underwent changes in the absence of protein synthesis de novo. These results suggest that there is a novel pathway for the action of RA that is independent of nuclear receptor-mediatedtrans-activation.

Mapping of the humanGSPT1 gene, a human homolog of the yeastGST1 gene, to chromosomal band 16p13.1

TheGSPT1 gene, a human homolog of the yeastGST1 gene (formerly namedGST1-Hs), was mapped on human chromosome 16p13.1 by a combination of nonradioactive in situ hybridization and Giemsa staining. Southern blot hybridization with a panel of human-rodent somatic cells confirmed the location of theGSPT1 gene on chromosome 16 and also showed the existence of a homologous gene on the X chromosome. A breakpoint for nonrandom chromosome rearrangements has been found in the region ofGSPT1 in patients with acute nonlymphocytic leukemia.

GENOMIC CLONING AND PARTIAL CHARACTERIZATION OF HUMAN CHYMOTRYPSINOGEN GENE

SummaryChymotrypsinogen is a principal precursor of pancreatic proteolytic enzymes. We previously isolated a cDNA clone for human prechymotrypsinogen from a human pancreatic cDNA library. In the present study, we used this cDNA sequences to isolate genomic DNA clones. Three overlapping cosmid clones spanning approximately 65-kb genomic sequences were isolated from a human cosmid library. The genomic DNA clones were characterized by restriction enzyme mapping and by hybridizing them to subfragments of the cDNA. The sequence tagged sites for human chymotrypsinogen gene were created by designing two oligonucleotides. Furthermore, the isolated genomic clones were confirmed to be localized on chromosome 16q23 by fluorescence in situ hybridization and G-banding analysis.

Generation of 19 STS markers that can be anchored at specific sites on human chromosome 21

Sequence-tagged sites (STSs) are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR) and can be used to construct long-range physical maps of chromosomal DNA. These STSs can be detected by PCR assays developed by reference to data obtained from the sequencing of restriction fragment length polymorphism-DNA markers for chromosome 21, which were derived from recombinant lamba-phage and plasmid clones made from DNA of a human-hamster hybrid cell line. In this report, we describe the generation of 19 new STSs that are specific for human chromosome 21.